Electrospinning of polyacrylonitrile was used to investigate the effect of horizontal vs. vertical fiber deposition methods and operating variables on the degree of nanofiber alignment. A high-speed camera and Fiji image analysis software were used to assess differences and changes in the degree of fiber parallelism, allowing for effective analysis of the impact of operating variables on fiber parallel alignment. The results show that the strength of the electric field affects the fibers to assume a parallel alignment. Too weak the electric field, the fibers are unable to form a straight bundle, but too strong a field results in chaotic fiber alignment. The distance between the collectors influences the angle distribution of the fiber parallel alignment greatly. Comparing the effect of horizontal and vertical electrospinning on fiber alignment shows that horizontal results in a more optimal degree of parallel fiber alignment.